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Step-by-Step Examples

Computing Ki for a Competitive Enzyme Inhibitor

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A competitive enzyme inhibitor interferes with binding of substrate to enzyme so as to raise the Km value without
affecting Vmax. Competitive inhibition is overcome by increasing substrate concentration. A competitive inhibitor I
increases the “apparent” value of Km according to the relationship

Km

app

 [I ] 
= K m 1 +

 Ki 

where Ki is the dissociation constant for the enzyme·inhibitor complex. Ki is particularly useful for expressing the
potency of an inhibitor because, unlike IC50, it is independent of substrate concentration.
There are several graphical methods for estimating Ki, but the underlying experiment is the same—the
investigator generates substrate-velocity curves in the presence of various concentrations of inhibitor.

Increasing
[I]

v

[s]

1 Adapted from: Miller, J.R., GraphPad Prism Version 4.0 Step-by-Step Examples, GraphPad Software Inc., San
Diego CA, 2003. Step-by-Step Examples is one of four manuals included with Prism 4. All are available for
download as PDF files at www.graphpad.com. While the directions and figures match the Windows version of
Prism 4, all examples can be applied to Apple Macintosh systems with little adaptation. We encourage you to print
this article and read it at your computer, trying each step as you go. Before you start, use Prism’s View menu to
make sure that the Navigator and all optional toolbars are displayed on your computer.

2003 GraphPad Software, Inc. All rights reserved. GraphPad Prism is a registered trademark of GraphPad
Software, Inc. Use of the software is subject to the restrictions contained in the software license agreement.
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2 . Second. Ki estimation required linearizing transforms and replots. [I] changing with each curve in the family. Prism can perform a global curve fit. and Ki are intrinsic to the system and therefore common to all the curves. but Prism offers two improvements. finding the single best-fit estimates of Vmax. First. [S] and [I] are independent variables. The rate equation is v= Vmax [ S ]  [I ]  K m 1 +  + [S ] K i   In the family of substrate velocity curves. This article includes the following special techniques: ƒ Global (shared-parameter) curve fitting ƒ Fitting data to a user-defined equation—entering the equation and setting rules for initial values ƒ Constraining fitted parameters—designating parameters to be shared among multiple data sets and fixing parameters to values extracted from data tables ƒ Segmented (gapped) axes Entering and Graphing the Data In the Welcome dialog. Complete the data table as shown below. Km. But Vmax. while each Y column holds the velocity value measured at the inhibitor concentration shown in the heading for that column.Formerly. and Ki for all curves taken together. the kinetic constants are more quickly and reliably estimated directly from nonlinear regression. make the settings shown below. part of the function of replotting is replaced by global (shared-parameter) curve fitting. Km. The X column holds substrate concentrations.

Prism will show those units in the graph legend.For this example. The second line is the same as the velocity equation given earlier. then enter the two-line system shown: The first line defines an intermediate variable equal to the apparent Km as discussed earlier. µmol/min/mg protein for Vmax). Give your user-defined equation a name. verify that the Equation tab is selected and then choose More equations. In the Parameters: Nonlinear Regression (Curve Fit) dialog. and Prism will fit the resulting equation to each data set. We’ll fix that later. Click the yellow Graphs tab to display the graph. select [Enter your own equation]. v= Vmax [ S ]  [I ]  K m 1 +  + [S ] K i   3 . Km app  [I ]  = K m 1 +   Ki  The right-hand side of that equation will be substituted for “Kmapp” in the second line. Click Analyze. but simply extract the numeric portion of the column heading for its curve-fitting analysis. nM for Km and Ki. The units notations in the column headings above are useful but optional. In the list below. Most of the points are crowded at the left-hand side of the graph. In the Analyze Data dialog. choose Nonlinear regression (curve fit) from the Curves & regression list. The User-defined Equation dialog opens. and the units of velocity are µmol/min/mg protein. we’re assuming that the units of [S] and [I] are nM. Prism is indifferent to these units—it is for you to remember that data on Results sheets are in units that match your input data (thus.

That means that these settings will apply whenever we use this user-defined model. For example. Prism will read “AB” as a parameter with that name. but it’s good practice to always include it. not as the product of A and B. The settings are shown below. Note that we have just specified shared parameters and constants in the User-defined Equation dialog. Prism identifies and displays the equation parameters to be fitted by nonlinear regression. Prism allows implied multiplication. and Vmax are common to all data sets. 4 . The multiplication operator * in the first line isn’t strictly necessary. the apparent Km changes. but it must not lead to ambiguity. in the presence of inhibitor. Ki. Designating Global (Shared) Parameters Select the Default Constraints tab.after substituting “X” for the independent variable [S]. Remember that. You can also make these settings under the Constraints tab in the Parameters: Nonlinear Regression (Curve Fit) dialog. Km. Refer to the Prism User’s Guide for more information about the rules for writing user-defined equations. so select Shared value for all data sets for those parameters. Km is a global parameter. the settings will apply only to the analysis you’re running when you make the settings and will not be part of the equation definition. not Km itself. Leave this dialog open as you proceed to the next section. We must now tell Prism (a) what parameters to treat as “shared” among data sets and (b) what values to use for [I] in the case of each data set. In that case.

in that it will set [I] equal to the numerical portion of the Y column heading for that data set. select Data set constant (=column title). Column heading constants are a powerful feature that effectively allows you to have a second independent variable. Click OK twice to exit the Parameters dialogs and perform the curve fit. see the Step-by-Step Example “Fitting Data to User-Defined Equations”. Here is a partial view of the Results sheet: 5 . Completing the Curve Fit Select the Rules for Initial Values tab. This causes Prism to fit a different equation to each data set. Enter initial-values rules as shown below (for more on setting for initial values.Assigning Parameter Values from Y-Column Headings Next to “I” (see dialog above).

see the Step-by-Step Example “Analyzing Dose-Response Data”. You don’t have to use the same X values for each data set on your table. consider using a different distribution of substrate concentrations at different concentrations of inhibitor. we’ll segment the X axis to eliminate the space between X = 1050 and X = 9000 and to use different scales for each segment: 6 .The values for fitted (remember that I is constrained) parameters in all columns are the same because they are all shared parameters. and that has been done by fitting the parameters globally. choose Change… Analysis Parameters. For an example of using different X values. If you want to change any of the parameter settings and re-run the curve fit. 100 0 nM 3 nM 10 nM 30 nM 75 50 25 0 0 2500 5000 7500 10000 12500 We can address the crowding of points into the left-hand side of the graph by dividing the X axis into two segments. in an actual experiment. The object here is to get the best estimate for Ki. Placing a Gap in the X Axis In this section. The curves measured at higher inhibitor concentrations are not covered as uniformly by the data points. Graph of the Fitted Data Click on the yellow Graphs tab to display the graph. We wanted to keep the data table in this example simple.

nM 60 0 nM 3 nM 10 nM 30 nM 40 20 0 0 250 500 750 1000 10000 Concentration substrate (nM) Family of Substrate-Velocity Curves with Gapped (Segmented) X Axis See text for instructions on producing the gap. Double-click on the X axis to open the Format Axes dialog. The graph includes other formatting changes covered elsewhere in this book. A particularly tricky part may be to decide on the relative lengths of the segments (Length % of axis). Under Gaps and Direction.Velocity (µmol/min/mg) 80 [Inhibitor]. Now you must set range and tick options for each segment. these settings may require some experimentation. then set the range and tick options for the right segment as follows: For other graphs. Select Left from the list box labeled Segment. Set the range and tick options for the left segment as follows: Now select Right from the Segment list box. Make sure the X axis tab is selected. click 7 . choose to divide the axis into Two segments. An easy way to adjust this is to switch to the graph.

Prism then adjusts the Length % of axis setting automatically.to select either of the segments. 8 . then drag the end of that segment to adjust its length.